1. Field of the Invention
This invention pertains to a vaccine composition which provides immunity from clinical disease signs and/or infections caused by lentiviruses including but not limited to Equine Infectious Anemia Virus (EIAV), Human Immunodeficiency Virus (HIV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency (BIV) and Simian Immunodeficiency Virus (SIV) or any other similar lentivirus. More specifically, but without limitation hereto, the invention relates to an Equine Infectious Anemia Virus (EIAV) composition which provides immunity from clinical disease signs and/or infection with EIAV, and which composition allows diagnostic differentiation between vaccinated and non-vaccinated but exposed or diseased mammals, and which allows the vaccinated animal to test negative using a Coggins test or other similar test that detects p26-specific antibodies
2. Brief Description of the Prior Art
Lentiviruses are a subfamily of retroviruses that cause persistent infection and chronic disease in numerous types of mammals including humans (HIV), equines (EIA), felines (FIV), bovines (BIV) and monkeys (SIV). All of the diseases are spread by blood transmission. EIAV causes persistent infection and chronic disease in horses world wide. With EIAV, the blood transmission occurs by biting flies and other insects carrying virus particles from one horse to another. The first cycle of disease (clinical episode) in an infected horse usually occurs within 42 days after exposure to the virus. This first cycle is usually referred to as the acute stage of EIA and is characterized by pyrexia, thrombocytopenia, anorexia, depression and high plasma viremia levels. Anemia is not usually detected at this stage. Resolution of this first febrile episode is normally observed after 1 to 5 days and occurs concomitantly with a dramatic drop in the amount of plasma-associated virus. Following the acute stage, some animals may remain clinically normal while others go on to experience multiple bouts of illness in which severe anemia may accompany pyrexia, thrombocytopenia, edema, and dramatic weight loss, and death. In instances of persistent infection by a lentivirus, as illustrated by EIAV, nucleotide sequence data has revealed a high mutation rate of the lentivirus genome as reported by Payne et al, Virology, 1987: 161, p. 321-331 which is incorporated herein by reference. With EIAV infections, it is generally thought that neutralizing antibodies aid in the selection of new antigenic virus variants during persistent infections. Also, with EIAV infections, serologically distinct variants of EIAV emerge possibly through immune selection pressure operating on random viral genome mutations. Without being bound to any particular theory, it is believed that horses that show no further clinical signs of disease have developed a mature immune response that can protect against the virus and its known mutations.
As a member of the lentivirus subfamily of retroviruses, EIAV is useful as a model for the pathogenicity, immunology, vaccinology, treatment and prevention of HIV. The disease is significant in its own right because horses that demonstrate exposure to EIAV as measured by testing for anitbodies in the blood (Coggins Test or similar p26 detecting test) are either required to be destroyed or strictly quarantined. As a result of the Coggins Test requirement and its broad use throughout the world, especially in testing performance horses that are transferred into and out of the United States, it is critical that any effective EIA vaccine not be able to seroconvert horses to a positive Coggins Test or to any other test that detects p26. Therefore, for vaccines useful in protecting against EIA, it is important to either delete all or part of the gene expressing p26 or block its expression by deleting regulator genes or inserting stop codons or foreign genes. It is expected that use of the methods described herein can provide vaccines for the other lentiviruses (HIV, FIV, BIV and SIV) that can elicit immune responses that are effective and that can be distinguished from viral infections.
As with other lentiviruses such as HIV, BIV, FIV and SIV, the genetic organization of EIAV classifies it as a complex retrovirus. The EIAV genome contains the canonical gag, pol, and env genes common to all retroviruses, and three accessory genes (S1, S2 and S3). The gag gene encodes the core proteins of the virus designated as Matrix Antigen (MA), Capsid Antigen (CA), Nucleocapsid (NC) and a protein designated p9. The env gene encodes the viral envelope proteins (gp90 and gp45). The pol gene encodes the enzymes that replicate the viral genome, designated as Deoxy UTPase (DU), Reverse Transcriptase (RT) and Integrase (IN). The S1 open reading frame (ORF) encodes the viral Tat protein, a transcription trans activator that acts on the viral long-terminal-repeat (LTR) promoter element to stimulate expression of all viral genes. The S3 ORF encodes the Rev protein, a post-transcriptional activator that acts by interacting with its target RNA sequence, named the Rev-responsive element (RRE), to regulate viral structural gene expression. The S2 gene is located in the pol-env intergenic region immediately following the second exon of Tat and overlapping the amino terminus of the Env protein (see FIG. 1). It encodes a 65 amino acid protein with a calculated molecular mass of 7.2 kDa. S2 appears to be synthesized in the late phase of the viral replication cycle by ribosomal leaky scanning of a tricistronic mRNA encoding Tat, S2 protein, and Env protein, respectively.
The gag-encoded Capsid Antigen (CA) or p26 protein comprises the capsid shell of the virion that is enclosed in the viral envelope and that contains the viral RNA genome. Homologous CA proteins are present in HIV, FIV, BIV and SIV and are also encoded by the respective gag genes. As noted above, detection of antibodies to the p26 antigen is the basis for the Coggins Test and certain other commercial tests used to diagnose EIA in horses. To be compatible with current regulatory guidelines, it is critical that any EIAV vaccine not stimulate seroconversion in these diagnostic assays based on detection of serum antibodies to EIAV p26. The p26 antigen is highly antigenic in that extremely small amounts of its presence in a vaccine can stimulate antibody responses and seroconversion in diagnostic assays. Attempts to extract or delete p26 antigen from a pool of EIAV have not been practical for vaccine production. Therefore, it would seem that one could eliminate it by deletion of the gag gene, a segment of the gag gene that interferes with the expression of p26 or deletion or inactivation of a control gene that regulates the expression of p26. However, it has been determined by the inventors that deletion of the gag gene or segments thereof produces an EIAV particle that is unable to replicate in vitro (tissue culture) or in vivo. Therefore, simply deleting or blocking expression of p26 makes growth of EIAV for vaccine production impractical if not impossible.
To provide protection from disease and protection from infection, envelope proteins (Env) are considered the proteins of choice, as these proteins are the predominant immune targets during infection. By protection from disease is meant that a mammal exposed to the virus does not demonstrate clinical signs (fever, lethargy, anemia, death, etc.), but does carry virus particles in its blood, which particles are detectable by a reverse transcriptase polymerase chain reaction test (RT-PCR). By protection from infection is meant that a mammal exposed to the virus does not demonstrate clinical signs of EIA and does not contain RT-PCR-detectable virus particles in blood. The major envelope proteins of EIAV are gp90 and gp45. These are proposed as the protective antigens or protective components of EIAV. By the term protective components is meant antigens from that produce either protection from disease or protection from infection as indicated above. It is therefore important that any effective lentivirus vaccine contain amounts of the lentiviral Env proteins (such as gp 120, gp90 or gp45) effective to protect mammals from disease caused by the lentivirus. The protective components from EIAV include but are not limited to gp90 and gp45. The Capsid Antigen (p26) is not a protective component of EIAV and, because of its ability to stimulate a significant antibody response, the vaccine of the present invention preferably lacks the ability to stimulate p26 antibodies in an equid.
It would seem obvious to prepare a vaccine by purifying out the Env proteins, especially gp90 and gp45 for EIAV. Indeed, vaccines comprising preparation from which gp90 and gp45 have been purified out of the EIAV have been attempted with extremely limited success. Issel et al (J. Virol. June 1992, p 3398-3408) reports that a gp90/gp45 vaccine protected ponies from infection caused by homologous EIAV (the subunits were derived from the same EIAV strain as was used for challenge). However, these subunit-containing vaccines did not protect horses from either disease or infection when challenged with a heterologous EIAV strain. In fact, the latter produced enhanced disease signs. The enhancement of disease by the subunit EIAV vaccine corroborates findings with SIV and FIV subunit vaccines that appear to enhance disease post challenge. Issel et al (ibid) concludes that perfecting a subunit vaccine for lentiviruses (e.g., HIV, FIV, EIA, BIV and SIV) poses a significant challenge because of the subunit enhancement effect.
Issel, et al (ibid) also reports the prevention of infection by a whole-virus EIAV vaccine. However, vaccination of horses with this vaccine produces horses that are Coggins Test positive (p26 positive). As mentioned previously, due to the eradication program in effect in the U.S., horses testing positive for p26 are either euthanized or strictly quarantined. Additionally, the amount of virus included in said vaccine was 1 milligram, an amount not commercially feasible. Therefore, this whole-virus vaccine is not compatible with regulatory requirements or commercialization.
A donkey virus vaccine has been in use by the Chinese for more than 20 years. This vaccine was developed by using total EIAV genetic material from donkey leukocyte attenuated EIAV infected cells and ribonucleic acid from virus in peripheral blood of donkey-adapted EIAV from infected donkeys (see Xinhua News Agency, May 6, 1999). As would be expected, this vaccine produces a p26 positive response (Coggin""s Test positive) in vaccinated horses or other vaccinated equids. Such a vaccine is not acceptable in those countries where equids are tested by Coggins assays or other p26-specific antibody tests. In addition, numerous countries will not accept live vaccines for veterinary applications.
Since there has been no effective and safe method for immunizing mammals against disease or infection caused by lentiviruses, particularly equines against EIA, and since lentivirus diseases, especially HIV, FIV and EIA are such a wide-spread and significant diseases world-wide, there remains a long-felt need to prepare such a vaccine.
The vaccine of this invention provides a successful vaccine composition that effectively and safely immunizes mammals from diseases caused by lentiviruses. The vaccine of the present invention protects equines from EIA wherein vaccinated equines can be differentiated from wild-type infected equines, which does not convert said equines to Coggins Test positive and which does not replicate in vivo. It is fully envisioned that the vaccines taught by the present invention can be used for production of any lentivirus vaccines, including vaccines for HIV, FIV, BIV and SIV.